United States

Department of

Agriculture

Animal and Plant

Health Inspection

Service

Veterinary Services

Epidemiologic and Other Analyses of 

Avian Influenza Affected Poultry Flocks

February 2019 Report
Epidemiologic and Other Analyses of LPAI Affected Poultry Flocks February 1, 2019

Suggested bibliographic citation for this report: 

USDA-APHIS (2019). Epidemiologic and other analyses of avian influenza affected poultry flocks: 

February, 2019 Report. United States Department of Agriculture: Animal and Plant Health 

Inspection Service: Veterinary Services: Center for Epidemiology and Animal Health. Fort 

Collins, CO. April 2019. 72 pgs.

For more information, contact: 

Amy Delgado, DVM PhD

Director, Monitoring and Modeling

USDA:APHIS:Veterinary Services

Center for Epidemiology and Animal Health

2150 Centre Avenue, Bldg. B-2E7, Fort Collins, CO, 80526-8117

Phone (970) 494-7256 | Mobile (970) 237-0857 | Fax (970) 494-7228

Email: Amy.H.Delgado@aphis.usda.gov

Web: http://www.aphis.usda.gov/animal-health/ceah

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Contents

I. Executive Summary ....................................................................................................................1

II. Introduction ...............................................................................................................................2

Description of Outbreak .......................................................................................................................3

III. Epidemiologic Study to Investigate the H5N2 Virus in Commercial Poultry in Minnesota ..............5

Case Series............................................................................................................................................5

IV. Estimating the Time of H5N2 LPAI Infection in Commercial Meat-type Turkey Flocks using 

Diagnostic Test Results...............................................................................................................7

Summary...............................................................................................................................................7

Methods Overview................................................................................................................................8

Summary of Sampling Data...................................................................................................................8

Overview of Modeling Approach..........................................................................................................9

Results.................................................................................................................................................10

Discussion ...........................................................................................................................................16

V. Phylogenetic Analysis and Diagnostics.......................................................................................18

Phylogenetic Analysis and Diagnostics...............................................................................................18

North American H5N2 LPAI from turkeys (AM H5N2 2018)...............................................................18

Comparison to Other Viruses/Lineages..............................................................................................20

Public Health Aspects..........................................................................................................................20

Diagnostics and Characterization for Influenza A Viruses..................................................................20

General Information ...........................................................................................................................21

VI. Waterfowl Surveillance.............................................................................................................21

VII. Using geospatial methods to measure the relative risk of environmental persistence of avian 

influenza virus in Minnesota.....................................................................................................26

Summary.............................................................................................................................................26

Model Inputs and Development.........................................................................................................26

Model Execution .................................................................................................................................27

Results and Discussion........................................................................................................................28

VIII. References ...............................................................................................................................34

IX. Acknowledgements..................................................................................................................37

Appendix A: Case Series Questionnaire.............................................................................................38

Appendix B: Model Parameter Estimation ........................................................................................52

Barn-level LPAI diagnostic test data ...................................................................................................52
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Sensitivity Analysis: Number of Swabs in rRT-PCR Pooled Samples...................................................68

Stochastic Disease Transmission Model Details.................................................................................68

Estimation of Transmission Model Parameters..................................................................................69

Appendix C: Data Factors and Processing for Geospatial Analysis Predicting Avian Influenza Virus 

Persistence in the Environment.................................................................................................71

Data Preparation.................................................................................................................................71

Predictive Factors and Data Sources: .................................................................................................71

List of Figures and Tables

Figure 1. Counties with confirmed findings of H5N2 LPAI between October and November 2018. .........................4

Figure 2. Likelihood of obtaining the test result on the date it was observed in the data for a given day of 

introduction for individual rRT-PCR test results, individual AGID test results, and the overall likelihood of the 

combined test results estimated from the Stearns 1/Barn 2 diagnostic test data. .............................................14

Figure 3. Likelihood of obtaining the test result on the date it was observed in the data for a given day of 

introduction for individual rRT-PCR test results, individual ELISA test results, and the overall likelihood of the 

combined test results estimated from the Stearns 4/Barn 1 diagnostic test data. .............................................15

Figure 4. Contour plot of the likelihood of observing the diagnostic test results for Stearns 1/Barn2 given 

different adequate contact rate and date of virus introduction pairs. ................................................................15

Figure 5. Contour plot of the likelihood of observing the diagnostic test results for Stearns 4/Barn1 given 

different adequate contact rate and date of virus introduction pairs. ................................................................16

Figure 6. a) Phylogenetic analysis by gene for viruses from Kandiyohi (green) and Stearns (purple) counties; b) 

concatenated analysis of common genes (HA and NA) of viruses from Kandiyohi (green), Stearns (purple), and 

Canadian wild birds (blue)....................................................................................................................................19

Figure 7. The four primary North American waterfowl flyways. .............................................................................22

Figure 8. Sampling locations for avian influenza virus A in wild birds from March – October 2018. ......................23

Figure 9. Total samples collected by month between March and December, 2018. ..............................................24

Figure 10. Wild bird sample numbers by flyway between March and December, 2018. .......................................24

Figure 11. Wild bird matrix positive samples by month from March to October, 2018..........................................25

Figure 12. Risk (1 km resolution) of fall (September 1 – November 30) environmental persistence of AIV in 

Minnesota and LPAI-affected counties (October 20 – November 14, 2018). ......................................................28

Figure 13. County level AIV environmental persistence risk in Minnesota in fall (September 1 – November 30) 

and LPAI-affected counties (October 20 – November 14, 2018)..........................................................................30

Figure 14. Number of commercial poultry operations, counties with high fall season AIV environmental 

persistence risk, and LPAI-affected counties (October 20 – November 14, 2018)...............................................31

Figure 15. Number of backyard poultry operations, counties with high fall season AIV environmental persistence 

risk, and LPAI-affected counties (October 20 – November 14, 2018). .................................................................32
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Figure 16. 3km and 10km buffer zones around an individual commercial poultry operation (not shown) in the 

recent Minnesota LPAI outbreak (October 20 – November 14, 2018). Buffers are overlaid with the 1km AIV 

environmental persistence risk model. ................................................................................................................33

Table 1. Location, production type, and confirmation date of flocks infected by LPAI H5N2. ..................................3

Table 2. Characteristics of H5N2 LPAI infected commercial poultry farms. ..............................................................6

Table 3. Summary of surveillance protocols and test results from a barn on an LPAI-infected commercial meattype turkey premises (Stearns 1) in Minnesota. ....................................................................................................8

Table 4. Summary of surveillance protocols and test results from a barn on an LPAI infected commercial meattype turkey premises (Stearns 4) in Minnesota. ....................................................................................................9

Table 5. Mostly likely date of LPAI virus introduction (95% CI) for the barns that tested positive during the 2018 

H5N2 outbreak in Minnesota, estimated from rRT-PCR and serology diagnostic testing data. ..........................12

Table 6. The number of days from the first 100% seropositive test result until the first rRT-PCR negative test 

result with no rRT-PCR positive test results following, and the number of days from the first 100% seropositive 

test result until the first rRT-PCR test...................................................................................................................13

Table 7. Predicted and observed dates when infectious birds were no longer present in the flock following test 

results of 10/10 seropositive and 1/1 rRT-PCR positive samples for the five barns in which this test result was 

observed...............................................................................................................................................................14

Table 8. Percent sequence similarity between H5N2 LPAI in MN turkeys to the highly similar wild bird viruses 

from Canada supports independent introductions from a common source........................................................20

Table 9. 2018 H5 and H7 positive samples by state and date collected..................................................................25

Table 10. Environmental factors selected as model inputs, data sources, trends relative to AIV survival, and 

rationale for model inclusion. ..............................................................................................................................27

Table 11. Environmental factors, layer values, and PA add-in query expression weights used to develop the final 

predictive model for the persistence of avian influenza virus in the environment. ............................................27

Table 12. PA model values and reclassification into OIE risk categories. ................................................................29

Table 13. County-level AIV environmental persistence risk reclassification scheme. .............................................30

Table 14. Summary of surveillance protocols and test results from the LPAI infected barns on Kandiyohi 1. .......52

Table 15. Summary of surveillance protocols and test results from the LPAI infected barns on Kandiyohi 2. .......55

Table 16. Summary of surveillance protocols and test results from the LPAI infected barns on Kandiyohi 3. .......57

Table 17. Summary of surveillance protocols and test results from the LPAI infected barns on Kandiyohi 4. .......62

Table 18. Summary of surveillance protocols and test results from LPAI infected barns on Stearns 1. The testing 

summary for Barn 2 is included in the main report. ............................................................................................64

Table 19. Summary of surveillance protocols and test results from the LPAI infected barn on Stearns 2..............64

Table 20. Summary of surveillance protocols and test results from the LPAI infected barns on Stearns 3. ...........65

Table 21. Summary of surveillance protocols and test results from LPAI infected barns on Stearns 4. The testing 

summary for Barn 1 is provided in the main report.............................................................................................67
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Table 22. Most likely date of introduction (95% CI) for the barns that tested positive on Stearns 3 and Kandiyohi 

4 during the 2018 H5N2 outbreak in Minnesota considering rRT-PCR pooled samples of size 11 and 10 swabs.

..............................................................................................................................................................................68

Table 23. Parameter estimates for the LPAI transmission model for turkey barns. ................................................69

Table 24. Common and scientific names of dabbling duck species that were included in the wild bird presence 

model....................................................................................................................................................................72
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I. Executive Summary

An outbreak of low pathogenic avian influenza (LPAI) was detected in October-November, 2018, 

in the State of Minnesota. The index flock was detected through avian influenza surveillance 

testing as part of an industry-driven enhanced sampling surveillance which has occurred in the 

spring and fall in certain counties in central Minnesota since the 2015 highly pathogenic avian 

influenza outbreak. The first detection was on a commercial meat tom turkey flock in Kandiyohi 

County. Laboratory testing by National Veterinary Services Laboratories (NVSL) confirmed H5N2 

LPAI of North American wild bird lineage. Subsequently, between October and November, 2018, 

H5N2 LPAI virus was detected on a total of eight commercial turkey premises in Kandiyohi and 

Stearns counties in Minnesota. Controlled marketing was completed for poultry on all infected 

premises in accordance with Minnesota’s Initial State Response and Containment Plan and 

USDA-APHIS-VS LPAI Guidance Documents. 

Following response activities, a series of epidemiologic and genetic investigations were initiated 

to better understand virus introduction and transmission. These investigations were a 

collaboration between the poultry industry, the State of Minnesota Board of Animal Health, and 

the United States Department of Agriculture Animal and Plant Health Inspection Service (USDAAPHIS). This is a report of findings available to date.

The epidemiologic investigation focused on factors and management practices which have been 

implicated as risk factors for infection in previous avian influenza outbreaks. Similarly, 

transmission through the movement of fomites such as people, vehicles, and equipment 

between farms, were likely. Additionally, breaches in biosecurity, for example, contact with wild 

birds, may have had a role in introduction and transmission. Epidemiologic, phylogenetic, and 

estimated time of introduction data supports independent introductions from a common source 

in the two counties, with limited subsequent spread between farms likely occurring. All 

recovered viruses have been characterized as North American wild bird lineage H5N2 LPAI, and 

representative viruses have an intravenous pathogenicity index (IVPI) of zero as defined by OIE. 

Waterfowl sampling in the U.S. from 1 March to 31 October 2018 was opportunistic and limited 

to 8 states with the majority of samples collected from live-captured birds in the months of 

August and September. Only one H5 virus was successfully isolated during this timeframe. Wild 

bird data shared from the National Center for Foreign Animal Diseases (NCFAD, Winnipeg, 

Canada), revealed highly similar wild bird viruses collected in July 2018 from the 

Central/Mississippi Flyway.

Diagnostic testing data was used to estimate the most likely date of virus introduction for all the 

barns that tested positive by rRT-PCR on the eight LPAI H5N2 virus infected turkey premises. 

These estimates helped industry veterinarians target their review of visitor logs and other 

investigation activities to identify possible pathways of introduction and to conduct tracebacks 

on likely exposures. Predictions from diagnostic test results were requested by some industry 

veterinarians during the outbreak to support operational planning decisions such as scheduling 
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birds for processing. The disease transmission simulation model was used to predict the time at 

which there were no infectious birds in the barn for a given set of diagnostic test results. In the 

current analysis, predictions were made for 5 barns, Kandiyohi 3/Barn 8, Stearns 2/Barn 1, 

Stearns 3/Barn 1, Stearns 3/Barn 2, and Stearns 4/Barn 4. The model predictions were 

consistent with the observed rRT-PCR interval for all 5 barns; however, the model predictions 

were conservative compared to the observed virus isolation interval, with the predicted interval 

being later than the observed interval in 3 out of 5 barns. The results of this analysis 

demonstrate the usefulness of diagnostic testing to better understand the behavior of LPAI in 

infected poultry flocks.

Previous epidemiologic investigations which employed sampling of wild birds surrounding 

infected farms have been unable to identify the virus in wild bird reservoirs, suggesting that the 

virus may have been shed and persisted in the environment prior to being introduced to a 

poultry farm. To explore this possibility, we identified areas in Minnesota at heightened risk for 

AIV environmental persistence during the fall migration season using geospatial methods. These 

results can be used to improve surveillance activities and to inform biosecurity practices and 

emergency preparedness efforts within Minnesota.

II. Introduction

In response to the H5N2 LPAI outbreak in commercial turkey operations in Kandiyohi and 

Stearns counties, Minnesota, USDA-APHIS and the Minnesota Board of Animal Health initiated 

epidemiologic and genetic investigations in an effort to better understand the risk factors 

associated with introduction of avian influenza into poultry flocks and subsequent transmission.

These investigations included the following:

• A field-based study of infected farms using data collected through site visits and 

interviews with farm personnel

• Analysis of barn-level mortality and diagnostic test data from the earliest affected 

commercial farm to estimate the date of virus introduction

• Virus phylogenetic analysis

• Summary of waterfowl surveillance from 1 March 2018 to 31 October 2018

• Geospatial estimation of the relative risks of environmental persistence of avian 

influenza viruses in the environment in Minnesota.

This report includes the results from those investigations, in an effort to provide producers, 

industry, and other stakeholders with current epidemiologic information.
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Description of Outbreak

On October 19, 2018, drinker biofilm samples1 were collected as part of the industry-driven 

enhanced sampling influenza surveillance. When these samples were determined to be nonnegative, oropharyngeal (OP) swab samples were collected as part of the follow-up testing to 

determine the status of the commercial meat-type turkey tom flock in Kandiyohi County, 

Minnesota. OP samples tested positive by H5 PCR at the Minnesota Poultry Testing Laboratory. 

The National Veterinary Services Laboratories (NVSL) confirmed H5N2 LPAI on October 20, 2018. 

A total of eight commercial turkey flocks were confirmed infected by NVSL with H5N2 LPAI in 

Kandiyohi and Stearns counties, Minnesota, between October 20, 2018 and November 16, 2018

(Table 1, Figure 1). Farms were detected through enhanced surveillance, pre-market testing, or 

as a result of surveillance testing in zones established around infected premises. Clinical signs 

were not apparent in most flocks, however, respiratory signs were reported in two flocks. NVSL 

confirmed North American wild bird lineage H5N2 LPAI on all farms based upon partial HA/NA 

sequencing. Following detection, surveillance was immediately initiated for commercial and 

backyard premises located within a 10km zone around all infected farms and for 

epidemiologically linked premises. In the absence of severe clinical signs and additional 

laboratory results, Veterinary Services and the Cooperating State Agency agreed that poultry on 

the infected premises pursue controlled marketing in accordance with Minnesota’s Initial State 

Response and Containment Plan and USDA-APHIS-VS Guidance documents. Controlled 

marketing was completed for all flocks by January 17, 2019.

Table 1. Location, production type, and confirmation date of flocks infected by LPAI H5N2.

State County Production Type Confirmation Date

Minnesota Kandiyohi Commercial Turkey 20 October 2018

Minnesota Kandiyohi Commercial Turkey 5 November 2018

Minnesota Kandiyohi Commercial Turkey 13 November 2018

Minnesota Kandiyohi Commercial Turkey 16 November 2018

Minnesota Stearns Commercial Turkey 31 October 2018

Minnesota Stearns Commercial Turkey 2 November 2018

Minnesota Stearns Commercial Turkey 5 November 2018

Minnesota Stearns Commercial Turkey 5 November 2018

 1 While drinker biofilm samples are not considered an “official sample” per NPIP/NAHLN, this sample type has 

been shown to allow detection of avian influenza RNA up to 24 hours ahead of animal samples. Approved 

laboratories accepting this sample type for testing are instructed to request appropriate samples from the flock 

immediately following any non-negative result to determine the virus status of the flock.
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Figure 1. Counties with confirmed findings of H5N2 LPAI between October and November 2018.
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III. Epidemiologic Study to Investigate the H5N2 Virus in Commercial 

Poultry in Minnesota

Case Series

In collaboration with State Animal Health Officials, USDA-APHIS conducted a case-series study of 

the H5N2 LPAI infected commercial turkey operations. A questionnaire was administered to

individual(s) on each farm most familiar with the farm’s management and operations. Questions 

focused on the three week period prior to the detection of LPAI (Appendix A). The purpose of 

this study was to generate hypotheses for potential risk factors for infection with LPAI based on 

descriptive information about farm characteristics and management practices.

Transmission of virus in previous outbreaks of avian influenza in the United States has been 

attributed to the movement of live and dead birds, transportation of manure, equipment 

sharing, and contaminated feed trucks, vehicles, water, and people (McQuiston et al., 2005, 

Halvorson, 2009, Garber et al., 2016). Results of the questionnaire, focusing on risk factors 

identified in previous outbreaks, are summarized below and in Table 2.

• Among the 8 infected farms, the median farm capacity was 53,000 birds, with a range 

from 35,000 to 174,000 birds. The number of barns on farms ranged from 3 to 10 barns, 

with an average barn capacity of 10,750 birds (range: 7,770 – 37,000).

• Most farms (5/8) were single age, and 3/8 were multi-age. Commercial toms were 

raised on 5/8 farms and commercial hens were raised on 3/8 farms. 50% of farms were 

grow only, 50% were brood and grow, and there were no breeders.

• The majority of farms (6/8) reported no clinical signs, however, two farms did report 

respiratory signs.

• A median reported distance to the closest body of water was 665 yards (range: 0-3,500 

yards). Water body types were varied and included pond, lake, stream, river, holding pit, 

slough, and man-made ditch. All farms reported that the primary water source for the 

farm was a well. Most respondents (7/8) reported that water is treated prior to delivery 

to poultry.

• Farms reported a median of two employees (range: 1-3).

• The most frequently reported type of visitor was feed truck delivery. Other visitor types 

reported included service person, veterinarian, and feed consultant. 

• Nearly all farms reported seeing wild birds around the farm (including on adjacent 

habitats away from facilities and equipment, on farm but not in barns, and on farm and 

sometimes in the barns). Bird types most commonly reported were gulls and small 

perching birds.
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Results showed that some factors and management practices were shared across infected 

farms, however, the significance of these similarities is difficult to interpret given the small 

number of infected farms and the study design. When considered in conjunction with 

knowledge of practices and risk factors from previous outbreaks, this information may provide 

insights into trends of management practices over time and elucidate opportunities to 

implement additional mitigations in the future.

Table 2. Characteristics of H5N2 LPAI infected commercial poultry farms.

Characteristic Level or response n

Barn ventilation type Curtain sided 6/8

Environmental control 0/8

Side doors 2/8

Other 2/8

Workers are employed by other poultry 

operations

Yes 0/8

Family members are employed by other 

poultry operations or processing plants

Yes 0/8

Workers are restricted from contact with 

backyard poultry

Yes 8/8

Equipment sharing: Which equipment is 

farm specific (owned solely by this farm and 

not used on other farms?

Company vehicles/trailers Yes 5/8

Feed trucks Yes 2/8

Gates/panels Yes 5/8

Lawn mowers Yes 4/8

Live haul loaders Yes 1/8

Poult trailers Yes 3/8

Preloaders Yes 2/5

Pressure sprayers/ washers Yes 4/8

Skid-steer loaders Yes 4/8

Tillers Yes 4/7

Trucks Yes 4/8

Litter shed is present on farm Yes 4/8

Farm does partial cleanouts Yes 2/8

Litter disposal site On farm 0/8

Taken offsite 6/8

Both on farm and taken offsite 2/8

Dead bird disposal method On farm: burial 

pit/incinerator/composted/other

5/8

Off farm: landfill/rendering/other 4/8

Off farm: disposal performed by 

owner/employee/other

0/7

Type of carcass bin Covered 2/5

Uncovered 3/5
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IV. Estimating the Time of H5N2 LPAI Infection in Commercial Meattype Turkey Flocks using Diagnostic Test Results 

Summary

Determining the time of LPAI virus introduction in a flock is an important part of outbreak 

investigations. By narrowing the time window of possible virus introduction, we can better 

identify the potential routes of virus introduction and enhance our understanding of the pattern 

of disease spread. In this analysis, diagnostic testing data was used to estimate the most likely 

date of virus introduction for all the barns that tested positive by rRT-PCR on the eight LPAI 

H5N2 virus infected turkey premises in Minnesota in 2018. The analysis was performed using a 

simulation-based method in which the likelihood of observing the diagnostic test results was 

estimated from a within-house disease transmission model for various candidate times of 

exposure.

Using this method, Kandiyohi 1 and Stearns 1 were estimated to have been the first premises 

infected with a most likely date of introduction of October 7th. Stearns 2 was estimated to have 

been infected only shortly afterwards, on October 8th. The most likely dates of virus 

introduction estimated for the subsequent premises was October 19th for Kandiyohi 2, October 

26th for Stearns 3, and October 28th for both Stearns 4 and Kandiyohi 4. The last premises 

estimated to have been infected was Kandiyohi 3 on November 6th. The last barn estimated to 

have been infected was Kandiyohi 4/Barn 4, with a likely date of virus introduction of November 

23rd. These estimates helped industry veterinarians target their review of visitor logs and other 

activity on the infected premises to identify possible pathways of introduction and to conduct 

tracebacks on likely exposures. 

Predictions from diagnostic test results were requested by some industry veterinarians during 

the outbreak to support operational planning decisions such as scheduling birds for processing. 

The disease transmission simulation model was used to predict the time at which there were no 

infectious birds in the barn based on diagnostic test results. As an example, predictions were 

made based on a test result of 10/10 seropositive serum samples and 1/1 rRT-PCR positive 

samples with the samples for both tests taken on the same day. This test result was observed in 

5 barns during the outbreak, including Kandiyohi 3/Barn 8, Stearns 2/Barn 1, Stearns 3/Barn1, 

Stearns 3/Barn 2, and Stearns 4/Barn 4. For model validation, the predicted time range was 

compared to two different observed intervals in the diagnostic testing data, the time until 

negative rRT-PCR test results were obtained and the time until virus isolation was not successful. 

The model predictions were consistent with the observed rRT-PCR interval for all 5 barns. The 

model predictions were conservative with respect to negative virus isolation results, with the 

predicted time to stop shedding being later than the observed interval in 3 out of 5 barns.

The results of this analysis are subject to some uncertainty due in part to a lack of information 

from infection and transmission studies with the LPAI H5N2 strain. Proxy H5 and H7 LPAI strains 

were used instead in the transmission simulation model. Furthermore, the time between 

diagnostic tests was in some cases several days, which can also introduce uncertainty into the 

results. Nevertheless, the results of this analysis demonstrate the usefulness of diagnostic 

testing to better understand the behavior of LPAI in infected poultry flocks.
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Methods Overview

Summary of Sampling Data 

The time of virus introduction was estimated for each barn that tested positive by rRT-PCR on 

the eight LPAI virus infected meat-type turkey premises in Minnesota. In total, 33 barns tested 

positive across the eight premises. The data consisted of the results for oropharyngeal swabs 

tested by rRT-PCR and serum samples tested by AGID or ELISA for each barn. There is some 

uncertainty in the number of swabs in each rRT-PCR pooled sample. For the purposes of this 

analysis, each rRT-PCR pool was assumed to consist of 11 swab samples. A sensitivity analysis 

performed assuming 10 swabs per pool produced minor differences in the results, suggesting 

the results are robust to this change in the pool size. The results of the sensitivity analysis are 

given in Appendix B. A summary of the testing dates, type of testing, amount of sampling 

performed, and test results are given for two barns on two different premises in Table 3 and 

Table 4. The testing schedules for the other 31 barns are provided in Appendix B.

Table 3. Summary of surveillance protocols and test results from a barn on an LPAI-infected commercial 

meat-type turkey premises (Stearns 1) in Minnesota.

Stearns 1: Barn 2

Date Test type Samplea Test result

Average (+) Ct 

value/ELISA 

titer

10/30/2018 rRT-PCR 2 pooled samples 2/2 positive 19

10/30/2018 AGID 10 serum samples 7/10 positive NA

11/05/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/08/2018 rRT-PCR 3 pooled samples 1/3 positive 36

11/08/2018 ELISA 10 serum samples 10/10 positive 2233

11/12/2018 rRT-PCR 3 pooled samples 0/3 positive NA

a

Pooled samples consisted of 10 or 11 oropharyngeal swabs.
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Table 4 Summary of surveillance protocols and test results from a barn on an LPAI infected commercial

meat-type turkey premises (Stearns 4) in Minnesota.

Stearns 4: Barn 1

Date Test type Samplea Test Result

Average (+) Ct 

value/ELISA 

titer

11/02/2018 rRT-PCR 2 pooled samples 1/2 positive 37

11/06/2018 ELISA 10 serum samples 0/10 positive NA

11/12/2018 rRT-PCR 1 pooled sample 1/1 positive 21

11/12/2018 ELISA 10 serum samples 0/10 positive NA

11/19/2018 rRT-PCR 1 pooled sample 1/1 positive 32

11/19/2018 ELISA 10 serum samples 9/10 positive 2233

11/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/03/2018 rRT-PCR 1 pooled sample 0/1 positive NA

a

Pooled samples consisted of 10 or 11 oropharyngeal swabs.

Overview of Modeling Approach 

The possible dates of virus introduction for each barn were estimated using a simulation-based 

approach. The number of infectious and seroconverted birds over time were estimated from a 

within-house stochastic disease transmission model. These numbers were then used to estimate 

the likelihood of observing the test results for a given range of potential disease introduction 

dates. The most likely date of LPAI virus introduction and its 95% CI were estimated from the 

overall combined likelihood values for all the observed test results. 

The stochastic disease transmission model was parameterized specifically for turkeys infected 

with LPAI virus. Distributions for the length of the latent and infectious periods at the bird level 

were estimated from inoculation studies in which turkeys were infected with H5 or H7 LPAI virus 

strains using a maximum likelihood approach (Comin et al, 2011; Iqbal et al, 2012; PantinJackwood et al., 2017; Pillai et al., 2010; Saenz et al., 2012; Spackman et al., 2010). The same 

approach was used to estimate a distribution for the length of time from infection until 

seroconversion. However, due limited data on H5 or H7 LPAI virus strains, inoculation studies 

involving any LPAI virus strain were included in the estimation of the time to seroconversion 

distribution (Dundon et al., 2007; Homme et al., 1970; Morales, 2008; Preskenis, 2010). Detailed 

information on model parameters is provided in Appendix B.

The disease transmission model was also used to predict the time at which there were no 

infectious birds in the barn given a set of available diagnostic test results. For model validation, 

the predicted time until no infectious birds was compared to both the observed time period to 

obtain negative rRT-PCR test results and the observed time period until virus could not be 

isolated from the rRT-PCR sample. The observed range of dates from the rRT-PCR test results 

consisted of the date of the last rRT-PCR positive result and the date of the following rRT-PCR 
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negative. The range of dates from the virus isolation results consisted of the date of the last 

instance where virus could be isolated from an rRT-PCR sample and the following date when 

virus could not be isolated from an rRT-PCR sample. The test results considered consisted of 

10/10 seropositive serum samples and 1/1 positive pooled tracheal swab sample (up to 11 

swabs per pooled sample) tested by rRT-PCR, with both the serum and oropharyngeal swab 

samples taken on the same day. Predictions were made for the 5 barns in which these test 

results were observed during the outbreak.

Results

The estimated most likely date of virus introduction (95% CI) for each barn ranged from 7 

October to 23 November 2018 (Table 5). Two barns were estimated to have been infected on 7 

October (Kandiyohi 1/Barn 2 and Stearns 1/Barn 3). Stearns 2/Barn 1 was estimated to have 

been infected next with a likely date of virus introduction only one day later, on 8 October. Next, 

Kandiyohi 2 was estimated to have been infected on 19 October. Stearns 3 was estimated to 

have been infected on 26 October, while Kandiyohi 4 and Stearns 4 were estimated to have 

been infected on 28 October. The premises estimated to have the latest date of virus 

introduction was Kandiyohi 3, which was estimated to have been infected on 6 November. 

However, the barn with the latest date of virus introduction was Kandiyohi4/Barn1, which was 

estimated to have been infected on 23 November.

The estimates for the time of virus introduction in barns on a single premises were in some 

cases quite variable. For example, the number of days between the earliest and latest most 

likely date of virus introduction for houses on Kandiyohi 4 was 26 days, the largest difference for 

any premises. In contrast, the difference between the first and last infected barns was estimated 

to be only 7 days on Kandiyohi 2 and Stearns 1. The average difference between the times of the 

first and last infected barns was estimated to be 13 days for the seven premises where more 

than one barn tested positive.

In order to visualize how well the actual test results align with the estimated introduction times, 

the likelihood of obtaining the test result on the date it was observed for a given time of virus 

introduction for individual rRT-PCR and serology tests, as well as the combined likelihood for all 

test events, are shown for Stearns1/Barn2 in Figure 2 and Stearns4/Barn1 in Figure 3. These 

figures illustrate how the likelihoods of individual test results vary across different possible 

introduction times and impact the overall likelihood considering all the test results.

Understanding the relationship between seroconversion and the time at which virus cannot be 

detected or isolated from an rRT-PCR test sample can improve our understanding of how LPAI 

spreads through flocks and the length of time until no infectious birds remain in the barn. In 12 

out of the 33 barns, at least one positive rRT-PCR test result was observed on or after the date 

when 100% of the serum samples tested positive. In comparison, virus isolation was successful

on or after the date of the 100% positive serum samples in only 4 barns. Table 6 provides the 

number of days between the first instance when 100% of the serum samples tested positive and 

the date when all rRT-PCR samples tested negative, as well as the number of days between the 

date of 100% positive serum sample results and the first date when virus could not be isolated. 

The median number of days between the 100% seropositive and negative rRT-PCR result was 4 
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days (0-15 days, 5-95% CI respectively). The median number of days between the 100% 

seropositive and negative virus isolation result was 0 days (0-7 days, 5-95% CI, respectively).

The average number of days between the estimated most likely date of virus introduction and 

the date of the first positive diagnostic test result across all the barns was 9 days. Similarly, the 

average number of days between the estimated most likely date of virus introduction and the 

first rRT-PCR negative test result, with all subsequent rRT-PCR samples also testing negative, was 

26 days. The average time between the estimated most likely date of virus introduction and first 

virus isolation negative test result was 20 days.

The relationship between the adequate contact rate parameter and the day of virus 

introduction is shown in Figure 4 for Stearns1/Barn2 and in Figure 5 for Stearns 4/Barn1. The 

figures display the likelihood for each evaluated contact rate and day of virus introduction pair. 

Lower contact rates represent slower within-house infection spread, which lead to earlier 

estimated virus introduction times. In both figures there is a well-defined high likelihood region 

for the day of virus introduction, which means both an upper and lower confidence bound could 

be obtained for the parameter estimate. In the case of the contact rate, only in Figure 5 is there 

a well-defined region with high likelihoods. In Figure 4, high likelihoods persist at the upper 

bound of the evaluated contact rate values. Figure 4 was the most commonly observed result in 

the barns, where a lower and upper bound could be estimated for the day of virus introduction, 

but only a lower bound could be estimated for the contact rate. 

The predicted and observed ranges for the date when infectious birds were no longer present in 

the barn following the test result of 10/10 seropositive serum samples and 1/1 positive samples 

by rRT-PCR are given in Table 7 for the five barns in which this test result was observed on a 

single day of testing. The predicted range compares favorably to the observed rRT-PCR test 

results with the 95% prediction interval (PI) overlapping with the observed range in all five

barns. Conversely, the PI and observed range from the virus isolation test results overlap in only 

two of the five barns.
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Table 5 Mostly likely date of LPAI virus introduction (95% CI) for the barns that tested positive during 

the 2018 H5N2 outbreak in Minnesota, estimated from rRT-PCR and serology diagnostic testing 

data.

Premises Most likely date of virus introduction (95% CI) Barn

Kandiyohi 1

October 7 (Sep 13 – Oct 09) Barn 2

October 18 (Oct 14 – Oct 19) Barn 1

October 23 (Oct 20 – Oct 24) Barn 4

October 24 (Oct 22 – Oct 25) Barn 3

Stearns 1

October 7 (Sep 22 – Oct 11) Barn 3

October 11 (Sep 29 – Oct 15) Barn 1

October 14 (Sep 27 – Oct 18) Barn 2

Stearns 2 October 8 (Sep 22 – Oct 13) Barn 1

Kandiyohi 2

October 19 (Oct 12 – Oct 22) Barn 1

October 19 (Oct 13 – Oct 23) Barn 5

October 24 (Oct 14 – Oct 28) Barn 3

October 25 (Oct 14 – Oct 28) Barn 2

October 26 (Oct 18 – Oct 30) Barn 4

Stearns 3

October 26 (Oct 19 – Oct 28) Barn 3

October 31 (Oct 27 – Nov 4) Barn 4

November 8 (Nov 2- Nov 10) Barn 1

November 8 (Nov 2 – Nov 10) Barn 2

Stearns 4

October 28 (Oct 14 – Oct 29) Barn 4

October 28 (Oct 18 – Oct 31) Barn 1

November 2 (Oct 27 – Nov 6) Barn 2

November 9 (Oct 27 – Nov 12) Barn 3

Kandiyohi 4

October 28 (Oct 19 – Nov 8) Barn 2

November 6 (Oct 23 – Nov 11) Barn 4

November 19 (Nov 10 – Nov 22) Barn 3

November 23 (Nov 15 – Nov 29) Barn 1

Kandiyohi 3

November 6 (Oct 31 – Nov 8) Barn 8

November 11 (Oct 31 – Nov 13) Barn 1

November 11 (Oct 31 – Nov 13) Barn 3

November 11 (Oct 31 – Nov 13) Barn 7

November 15 (Nov 9 – Nov 17) Barn 2

November 15 (Nov 9 – Nov 17) Barn 4

November 15 (Nov 9 – Nov 17) Barn 6

November 16 (Nov 9 – Nov 18) Barn 5
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Table 6 The number of days from the first 100% seropositive test result until the first rRT-PCR negative 

test result with no rRT-PCR positive test results following, and the number of days from the first 

100% seropositive test result until the first rRT-PCR test.2

Premises Barn

Days from the first 100% seropositive 

test result to first rRT-PCR negative, no 

subsequent positives

Days from the first 100% 

seropositive test result until the 

first negative virus isolation

Kandiyohi 1

Barn 1 11 days 0 days

Barn 2 15 days 8 days

Barn 3 14 days 0 days

Barn 4 14 days 0 days

Kandiyohi 2

Barn 1 4 days 4 days

Barn 2 3 days 0 days

Barn 3 11 days 4 days

Barn 4 4 days 0 days

Barn 5 4 days 0 days

Kandiyohi 3

Barn 1 0 days 0 days

Barn 2 0 days 0 days

Barn 3 0 days 0 days

Barn 4 0 days 0 days

Barn 5 0 days 0 days

Barn 6 0 days 0 days

Barn 7 0 days 0 days

Barn 8 5 days 0 days

Kandiyohi 4

Barn 1 0 days 0 days

Barn 2 0 days 0 days

Barn 3 NA NA

Barn 4 6 days 6 days

Stearns 1

Barn 1 0 days 0 days

Barn 2 4 days 0 days

Barn 3 6 days 0 days

Stearns 2 Barn 1 6 days 1 days

Stearns 3

Barn 1 8 days 7 days

Barn 2 7 days 7 days

Barn 3 0 days 0 days

Barn 4 0 days 0 days

Stearns 4

Barn 1 NA NA

Barn 2 NA NA

Barn 3 NA NA

Barn 4 21 days 0 days

 2 Cases where the negative rRT-PCR test or negative virus isolation result was observed prior to the 100% 

seropositive test result were set to zero. No 100% seropositive test result was observed in Barn 3 on Kandiyohi 4 

and Barns 1-3 on Stearns 4.
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Table 7 Predicted and observed dates when infectious birds were no longer present in the flock 

following test results of 10/10 seropositive and 1/1 rRT-PCR positive samples for the five barns in 

which this test result was observed.

Premises/

Barn

Sampling day 

for positive 

test results

Predicted most likely date no 

infectious birds in the flock 

(95% CI)

Observed date

rRT-PCR 

negative

Observed date 

virus isolation 

negative

Kandiyohi 3/

Barn 8

11/21/2018 12/02 (11/25 – 12/11) 11/21 – 11/26 11/15 – 11/21

Stearns 2/

Barn 1

10/31/2018 11/11 (11/03 – 11/21) 11/01 – 11/06 10/31 – 11/1

Stearns 3/

Barn 1

11/20/2018 12/01 (11/23 – 12/11) 11/27 – 11/28 11/20 – 11/27

Stearns 3/

Barn 2

11/20/2018 12/01 (11/23 – 12/11) 11/20 – 11/27 11/20 – 11/27

Stearns 4/

Barn 4

11/12/2018 11/23 (11/16 – 12/02) 11/26 – 12/03 11/2 – 11/11

Figure 2 Likelihood of obtaining the test result on the date it was observed in the data for a given day of 

introduction for individual rRT-PCR test results, individual AGID test results, and the overall 

likelihood of the combined test results estimated from the Stearns 1/Barn 2 diagnostic test data.
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Figure 3 Likelihood of obtaining the test result on the date it was observed in the data for a given day of 

introduction for individual rRT-PCR test results, individual ELISA test results, and the overall 

likelihood of the combined test results estimated from the Stearns 4/Barn 1 diagnostic test data.

Figure 4 Contour plot of the likelihood of observing the diagnostic test results for Stearns 1/Barn2 given 

different adequate contact rate and date of virus introduction pairs.
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Figure 5 Contour plot of the likelihood of observing the diagnostic test results for Stearns 4/Barn1 given 

different adequate contact rate and date of virus introduction pairs.

Discussion

Diagnostic test results can be used to estimate the day of LPAI virus introduction by providing 

insight into the stage of infection in the flock. Understanding of the transmission dynamics of 

the disease and of the flock must be considered as well; for example, caged birds would be 

expected to have a different transmission pattern than floor raised birds. Generally, where no 

seroconversion is detected in the face of virus detection suggest that the infection is in the 

earlier stages, when few birds have seroconverted. Conversely, if all serum samples test 

positive, then the infection is more likely to be in the later stages or no longer present in the 

flock. Where seropositivity (not all serum samples test positive) is incomplete, the rRT-PCR 

results from swabs aid in understanding the potential time of virus exposure. Baseline model 

results indicate that it may take 9 to 16 days post exposure for 50 percent of the flock to 

seroconvert depending on the rate of within house disease spread.

As can be observed in Table 5, Kandiyohi 1 and Stearns 1 had the earliest likely date of virus 

introduction of 7 October 2018. However, the presumptive index barn on Kandiyohi 1 had an 

earlier lower confidence bound of 13 September 2018 as compared to the presumptive index 

barn on Stearns 1, which had a lower confidence bound of 22 September 2018. Stearns 2 was 

estimated to have a highly similar most likely date of virus introduction of 8 October 2018. The 

similar estimated times of introductions for the index premises in these two counties is 

consistent with independent introductions from a common source, as also indicated by the 

phylogenetic data. The most likely date of virus introduction for the remaining premises, given 

in chronological order, was estimated to be 19 October for Kandiyohi 2, 26 October for Stearns 

3, 28 October for Kandiyohi 4 and Stearns 4, and 6 November for Kandiyohi 3. Narrowing the 
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time window of possible LPAI virus introduction can help in the identification of potential routes 

of virus introduction and improve the understanding of the pattern of disease spread. In this 

Minnesota outbreak, the model results were used by industry veterinarians to focus on 

potential epidemiological contacts during the estimated range of virus introduction times while 

screening out those occurring outside the window of potential virus introduction dates.

Diagnostic test results provide information on the flock status and suggests that such data could 

be used to make predictions in real-time during an outbreak to help assess risk and aid in 

decision-making. As an example, the time until no more infectious birds were present in the 

flock was predicted using the disease transmission model from a test result of 10/10 

seropositive and 1/1 rRT-PCR positive samples from five barns where this result was observed 

with the samples for both tests taken on the same day. The results in Table 7 indicate that the 

model provided reasonable predictions in all 5 barns for the observed range related to the rRTPCR test results. The model predictions were conservative with respect to negative virus 

isolation results with the predicted time to stop shedding being later than the observed interval 

in 3 out of 5 barns. The predictions may be improved by using a more informative contact rate 

distribution, which determines the rate of disease spread in the transmission model. 

In general, negative rRT-PCR and virus isolation results were observed within a short interval of 

observing a 100% seropositivity. However, viral RNA (e.g. rRT-PCR results) was detected in some 

barns up to 2 weeks after the 100% seropositive result. This is not an unexpected finding as 

experimental studies confirm that viral RNA may still be detected by rRT-PCR after 

seroconversion. In the current outbreak, no viable virus was recovered from any of the positive 

rRT-PCR samples collected between 3 and 13 days after the 100% seropositive result.

For rRT-PCR positive samples with Ct values above 35 (1 out of 31 rRT-PCR samples) virus was 

recovered only from one (Ct 37.7). Where the observed diagnostic test results, as well as the 

disease transmission simulation model, indicate that the flock would likely be at a later stage of 

infection several days after obtaining a 100% seropositive result, the prevalence of infectious 

birds would be lower. Further risk assessment is required to address how diagnostic testing data 

can be applied to support product movement decisions in conjunction with other considerations 

such as clinical presentation, environmental conditions, and applicable mitigations. Strainspecific experimental and outbreak data will further help reduce uncertainty and inform risk 

assessments.

Factors contributing to the uncertainty in the estimated time of virus introduction include 

uncertainty in model parameters such as the adequate contact rate, the inherent variability in 

disease spread dynamics, and the number and frequency of diagnostic tests results. Data from 

infection and transmission studies were not available on the LPAI H5N2 strain involved in the 

outbreak, leading to different H5 and H7 LPAI virus strains being used in the parameterization of 

the disease transmission model. The behavior of individual LPAI virus strains can differ 

substantially and the characteristics of the 2018 Minnesota H5N2 strain are uncertain. 

Nonetheless, the analysis presented here demonstrates the value of diagnostic testing data and 

its ability to provide information on disease dynamics within a poultry flock.
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V. Phylogenetic Analysis and Diagnostics

Phylogenetic Analysis and Diagnostics

North American H5N2 LPAI from turkeys (AM H5N2 2018) 

This section describes H5N2 LPAI from turkeys confirmed by the National Veterinary Services 

Laboratories (NVSL) in October 2018. The first detection in Kandiyohi County was based upon 

industry-driven enhanced sampling protocol which incorporates testing of drinker biofilm 

samples. A non-negative drinker biofilm result3 triggered collection of oropharyngeal (OP) swabs 

from turkeys on the premises which were tested the same day (19 October 2018) at MPTL with 

presumptive results by PCR for influenza A and subtype H5. Samples were hand carried to NVSL

the next day (20 October 2018), and NVSL confirmed H5N2 LPAI that evening by partial gene 

sequencing. Additional detections in Kandiyohi County followed. 

On 30 October 2018, non-negative results from routine blood samples collected for routine premovement testing in Stearns County triggered collection of OP swab samples from the animals. 

OP swab samples were collected and after samples were tested at MPTL they were sent to NVSL

for confirmation. An H5N2 LPAI virus was confirmed on the evening of 31 October 2018 by 

partial gene sequencing. Although the hemagglutinin (HA) and neuraminidase (NA) gene partial 

sequence was found to share 99% identity with the virus from Kandiyohi, the full genome data 

revealed differences across the internal genes and analysis showed that the HA genes clustered 

separately by county (Figure 6). 

All recovered viruses have been characterized as North American wild bird lineage H5N2 LPAI, 

and representative viruses have an intravenous pathogenicity index (IVPI) of zero as defined by 

OIE. The current phylogenetic analysis of viruses from the two counties supports independent 

introductions from a common source. Wild bird data shared from the National Center for 

Foreign Animal Diseases (NCFAD, Winnipeg, Canada), revealed highly similar wild bird viruses 

collected in July 2018 from the Central/Mississippi Flyway representing both introductions 

(Figure 6, Table 8). The lack of epidemiologic links between the counties further supports this 

finding. 

Weekly virus monitoring activities conducted in both counties during quarantine prior to control 

marketing revealed no changes in the HA gene cleavage site for any of the viruses obtained. The 

presence of an NA stalk deletion, which may be associated with virus adaptation in poultry, was 

observed from Kandiyohi 03 (refer to Figure 6b), and acquisition of an NA stalk deletion was 

noted from the fourth sampling (and the last from which virus was recovered) for Kandiyohi 01. 

NOTE: The outcomes of phylogenetic analysis should be interpreted in context of all available 

virus and epidemiologic information and should not be used directly to infer transmission.

 3 While drinker biofilm samples are not considered an “official sample” per NPIP/NAHLN, this sample type has 

been shown to allow detection of avian influenza RNA up to 24 hours ahead of animal samples. Approved 

laboratories accepting this sample type for testing are instructed to request appropriate samples from the flock 

immediately following any non-negative result to determine the virus status of the flock.
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Figure 6 a) Phylogenetic analysis by gene for viruses from Kandiyohi (green) and Stearns (purple) 

counties; b) concatenated analysis of common genes (HA and NA) of viruses from Kandiyohi4

(green), Stearns (purple), and Canadian wild birds5 (blue).

 4 NOTE: Kandiyohi 03 has a long branch due to the presence of a stalk deletion in the NA gene. 5 Courtesy of ML Killian NVSL, wild bird data from National Center for Foreign Animal Diseases, Winnipeg, Canada

a.

b.
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Table 8 Percent sequence similarity between H5N2 LPAI in MN turkeys to the highly similar wild bird 

viruses from Canada supports independent introductions from a common source.6

Comparison to Other Viruses/Lineages 

The H5N2 virus clusters with recent wild bird viruses and apart from other recent H5 poultry 

detections (2016 Missouri H5N1 LPAI, 2017 Wisconsin H5N2 LPAI). Highly similar viruses were 

identified from wild bird surveillance in Canada during July 2018 collected in the 

Central/Mississippi Flyway. 

Public Health Aspects 

To date, there have been no reports of the H5N2 LPAI 2018 virus infection in humans. The health 

of response workers and on-farm personnel was monitored at the state level. The virus sequences 

have been shared with CDC for analysis which indicated that the viruses to date lack key molecular 

features associated with human receptor binding, increased virulence, or transmission in 

mammals; no known markers of neuraminidase inhibitor (Oseltamivir) or polymerase inhibitor 

(baloxavir) resistance have been identified.

Diagnostics and Characterization for Influenza A Viruses 

The NVSL rapidly shares genetic and biological materials in collaboration with the Southeast 

Poultry Research Laboratory, the Influenza Division of the Centers for Disease Control and 

Prevention, USDA-APHIS Wildlife Services, as well as other key partners. Consensus data from 

whole genome sequencing are used to monitor the virus evolution and assess the risk to 

veterinary and public health based upon the presence/absence of specific amino acid 

substitutions or protein motifs. Analysis of sequence data includes phylogeny of all eight 

segments and determination of amino acid substitutions across the HA1 protein. Genetic data 

are also used to confirm that diagnostic assays are fit for purpose. In silico analysis confirmed 

high similarity between the H5N2 virus sequences and the relevant primers and probes used for 

the IAV and H5 diagnostic rRT-PCR tests.

 6 Courtesy of ML Killian NVSL, wild bird data from National Center for Foreign Animal Diseases, Winnipeg, Canada.

 y y 

Kandiyohi Co. to 

A/mallard/Sasketchewan/

OTH30-2/2018 (H5N2)

Stearns Co. to 

A/mallard/Sasketchewan/

OTH30-2/2018 (H5N2)

PB2 96% 95%

PB1 94% 95%

PA 99% 89%

HA 99% 99%

NP 99% 93%

NA 99% 99%

MP 99% 97%

NS (MALL) 99% 78%

NS (NOPI) 79% 99%
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General Information 

Avian influenza subtypes H5 and H7 are reportable worldwide because of their potential for 

mutation to high pathogenicity during replication in poultry. The presence of basic amino acids 

at the cleavage site contribute to the mutation from low to high pathogenicity. Mechanisms by 

which H5/H7 mutate from LPAI to HPAI include the gradual accumulation of basic amino acids 

(AA), insertion of repeated basic AA, and insertion of non-homologous genetic material (only 

reported for H7 viruses). 

Molecular diagnostic tests for influenza A virus (IAV) are used across the U.S. National Animal 

Health Laboratory Network (NAHLN). The most sensitive and specific tool for influenza A 

detection is the Type A-specific rRT-PCR, which targets at least the matrix gene (IAV-M); this is 

the primary surveillance tool used and provides a semi-quantitative result. The NAHLN tests 

samples first by the IAV-M test and further by the NAHLN H5 and H7 tests where IAV is detected. 

All poultry samples with a non-negative test result for IAV (serology or PCR) are forwarded to 

NVSL for confirmatory testing. The NVSL uses Sanger sequencing protocols to generate partial 

HA/NA gene sequence directly from the sample for subtype and pathotype determination, when 

sufficient viral RNA is present. Whole genome sequencing is conducted on all isolated viruses, 

and select viruses are further characterized by pathotype assay in specific pathogen-free 

chickens. 

NVSL confirms the virus HA and NA subtype through molecular sequencing and/or antibody 

subtyping, and the pathotype (LPAI vs HPAI); where no virus can be recovered nor sequence 

obtained directly from sample(s), the pathotype is determined by the clinical presentation of the 

flock compared to the USDA-APHIS HPAI case definition.

VI. Waterfowl Surveillance

Waterfowl are natural reservoir hosts for influenza A viruses (IAV; subtypes H1-H16), but not 

usually HPAI. Influenza A viruses in wild birds tend to circulate seasonally within migratory 

flyways, and subtype prevalence can wax/wane in multiyear cycles. Areas where birds from 

different flyways congregate provide opportunities for viruses to mix across flyways. 

Waterfowl migration in North America generally consists of north-south seasonal movements 

between breeding grounds and wintering areas. There are four major flyways in North America 

(Figure 7). These flyways are broadly defined corridors where the migratory paths of many 

species of interest tend to converge and are associated with major topographical features in 

North America, which also tend to be aligned along a north-south axis. The four North American 

flyways have areas of overlap and convergence, particularly at the north and south ends. Flyway

boundaries are defined administratively, and are not biologically fixed or sharply defined. 
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The U.S. National Surveillance Plan for Highly Pathogenic Avian Influenza in Wild Birds was 

implemented in 2015 to maximize our ability to detect IAV in wild waterfowl. Surveillance helps 

to: 1) understand how IAV is distributed in the United States, 2) detect the spread of IAV to new 

areas of concern, 3) monitor wild dabbling duck populations for introductions of novel viruses, 

and 4) estimate the apparent prevalence of IAVs of concern (e.g., Eurasian lineage H5 and H7). 

The surveillance plan targets areas with extensive mixing of wild bird populations and a history 

of IAV detection.

From March 1 to October 31, 2018, 1996 wild waterfowl and gulls were sampled and tested by 

rRT-PCR for avian influenza A infection. (Information on the sampling in January and February, 

2018, was summarized in the report Epidemiological and Other Analyses of Avian Influenza 

Affected Poultry Flocks, May 25, 2018)

Sampling was opportunistic and limited to eight states: Alaska, Oregon and Idaho in the Pacific 

Flyway; Colorado and North Dakota in the Central Flyway; Minnesota and Ohio in the Mississippi 

Flyway; and Connecticut in the Atlantic flyway (Figure 8, Figure 10). The majority of samples 

were collected from live-captured birds in the months of August and September (Figure 9). Gulls 

in North Dakota were sampled at landfill facilities in the course of management activities 

(n=206).

 Figure 7 The four primary North American waterfowl flyways.
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Avian influenza A was detected in a total of 602 samples, or 30% of the total. This is a high 

prevalence, and is skewed because, with limited resources for sampling, we targeted areas with 

historically high prevalence in the late summer, when the occurrence of AI in wild birds is 

highest. 

In spite of the high prevalence of Influenza A, only one H5 sample was successfully isolated

(Table 9). This was a blue-winged teal in Colorado with a low pathogenic H5N2 infection. One 

other bird was identified with a H5 infection, but no isolate was obtained. Six samples were 

presumptively positive for H7, but no isolates were obtained. All H5 and H7 samples were from 

dabbling ducks, sampled in August and September with the exceptions of one H5 being sampled 

on October 31 in Minnesota.

Figure 8 Sampling locations for avian influenza virus A in wild birds from March – October 2018.
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Figure 9 Total samples collected by month between March and December, 2018.

Figure 10 Wild bird sample numbers by flyway between March and December, 2018.

123

17

158

0 32

1022

630

14 0 0

0

200

400

600

800

1000

1200

2018 Samples, by month

1112

747

112

25

0

200

400

600

800

1000

1200

PAC CENT MISS ATLA

2018 Samples, by flyway
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Figure 11 Wild bird matrix positive samples by month from March to October, 2018.

Table 9 2018 H5 and H7 positive samples by state and date collected.

State Date Collected Subtype Species

Ohio Mar. 22 H7 LPAI Mallard

Alaska Aug. 3 H7 LPAI Mallard

Alaska Aug. 3 H7 LPAI Mallard

Alaska Aug. 3 H7 LPAI Mallard

Colorado Aug. 15 H7 LPAI American Green-winged Teal

Colorado Aug. 28 H7 LPAI Mallard

Colorado Sept. 17 H5N2 LPAI Blue-winged Teal

Minnesota Oct. 31 H5 LPAI Mallard

41

0 0 0 1

431

128

1

0

50

100

150

200

250

300

350

400

450

500

March April May June July August September October

2018 Matrix Positive Samples, by month 
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VII. Using geospatial methods to measure the relative risk of 

environmental persistence of avian influenza virus in Minnesota

Summary

Introduction of AIV into domestic poultry can be initiated by exposures to infectious wild birds 

or to virus surviving in the environment. This analysis aimed to define areas in Minnesota at 

heightened risk for AIV environmental persistence using geospatial methods. Environmental 

factors known to influence AIV survival were identified through an evaluation of the published 

literature, and corresponding data were located and downloaded from publicly available 

sources. Data layers were resampled to a 1km resolution and weighted based on their influence 

on virus survivability. Maps showing the relative risk of AIV persistence in the Minnesota 

environment were created from these layers using ESRI’s ArcGIS Predictive Analysis Tool. Five 

categories defined in the World Organization for Animal Health (OIE) Risk Assessment 

Guidelines were assigned to each 1km cell ranging from very low/negligible to very high. The 

results presented in this report focus on risk during the fall migration season (September 1 –

November 30) which completely encompasses the 2018 LPAI outbreak period from October 20 

through November 14. A majority filter was then employed to summarize risk for each county in 

Minnesota. Finally, maps were produced showing the number of commercial and backyard 

poultry premises in relation to highest risk counties. These results can be used to improve 

surveillance activities and to inform biosecurity practices and emergency preparedness efforts 

within Minnesota.

Model Inputs and Development

Factors known to influence AIV introduction and survival in the ambient environment, focusing 

on H5 and H7 subtypes, were identified through an evaluation of the peer-reviewed literature. 

This information was combined with an assessment of data availability and a total of five factors 

(water presence, water temperature, wetland cover, presence of wildlife refuges, and presence 

of wild birds) were selected as inputs to develop risk models. These factors, their corresponding 

data sources, trends relative to AIV survival, and rationale for model inclusion are summarized in 

Table 10. A detailed description of data processing for each factor is available in Appendix C.
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Table 10 Environmental factors selected as model inputs, data sources, trends relative to AIV survival, 

and rationale for model inclusion.

Model Execution 

Risk of environmental AIV persistence was determined using the Esri Predictive Analysis (PA) 

Tools Add-In within ArcGIS 10.6 (PA Add-In: http://appsforms.esri.com/products/download/). 

The PA Add-In is a collection of tools used to create models to predict the location of suitable 

sites based on input factors, in this study environmental or wild bird conditions. The PA tool was 

used to build a simple additive expression with environmental factors weighted based on their 

relative contribution to the persistence of AIV, as determined through subject matter expert 

(SME) consultation and literature review (Table 11).

The 1km risk model was then aggregated to a county level using a majority statistic tool in 

ArcMap. Additional analyses were performed to determine the proximity of LPAI-infected 

operations to moderate and high risk 1km cells within 3km and 10km.

Table 11 Environmental factors, layer values, and PA add-in query expression weights used to develop 

the final predictive model for the persistence of avian influenza virus in the environment.

Factor Layer Values Weighted PA Values

Wild Birds 1 = Presence 3

0 = Absence 0

Water Temperature 1 = < 10oC 3

2 = 10oC ≤ > 20oC 2

3 = ≥20oC 1

Water Presence 1 = Fresh water 3

0 = No water 0

Wetlands 1 = Presence 2

0 = Absence 0

Wildlife Refuges 1 = Presence 3

0 = Absence 0

Factor Data Source Trends with AIV Rationale

Water 

Presence

USGS Gap Analysis Program AIV particles survive well in water compared 

to air or other dry media

AIV survives in water more 

prominently than dry land

Water 

Temperature

USGS Moderate Resolution 

Imaging Spectroradiometer 

(MODIS) remote sensing 

data

Inverse association between persistence and 

increasing temperature: optimal 

temperatures are near freezing and suboptimal temperatures are 17-28 oC 

Temperature of water sources greatly 

impacts the rate of survival of AIV

Wild Bird 

Presence

Dabbling Duck Occurrence 

and Abundance model 

(USGS Patuxent Wildlife 

Research Center)

Wild birds are a main reservoir for AIV, and 

introduce the virus to their surrounding 

environment

When carrying the virus, wild birds 

deposit viral particles into water and 

other habitat locations

Wetland Cover United States Fish and 

Wildlife Service

Wetland cover provides ideal habitat for 

migratory birds, and contains aquatic zones 

where AIV can thrive for long periods 

Previous studies show that wetland 

areas are associated with AIV 

presence

Wildlife 

Refuges

United States Fish and 

Wildlife Service

Wildlife refuges are preservation zones of 

wild bird habitat

Refuges provide ideal habitat for AIV 

reservoir birds
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Results and Discussion

The raw model output values (ranging from 0 – 14) were reclassified by quantile to illustrate 

relative risk (OIE categories ranging from 0 – 5) (Figure 12). The quantile classification method 

distributes roughly the same number of 1km cells into each of the five risk categories. The five 

risk categories displayed correspond to the OIE Risk Assessment Guidelines for describing risk: 

negligible/very low, low, moderate, high, and extremely high risk (OIE, 2017). Reclassification 

followed the scheme outlined in Table 12.

Figure 12 Risk (1 km resolution) of fall (September 1 – November 30) environmental persistence of AIV 

in Minnesota and LPAI-affected counties (October 20 – November 14, 2018).
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Table 12 PA model values and reclassification into OIE risk categories.

Original model value Risk category (value)

0 Negligible/Very low risk (0)

1-2 Low risk (1)

3-4 Moderate risk (2)

5 High risk (3)

6-14 Very high risk (4)

The risk of environmental persistence of AIV derived from the 1km resolution models was then 

aggregated to the county level using the majority zonal statistic in ArcMap (Figure 13; 

http://desktop.arcgis.com/en/arcmap/10.3/tools/spatial-analyst-toolbox/zonal-statistics.htm). 

The majority zonal statistic computes the most frequently occurring original model value within 

each zone, in this case, the county. The resulting majority values for each county were 

reclassified into one of three risk categories following the scheme in Table 13. Out of 87 

counties in Minnesota, 61 counties, including those affected by LPAI in fall 2018 (Kandiyohi and 

Stearns), had low risk, 11 counties had moderate risk, and 15 counties had high risk.
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Figure 13 County level AIV environmental persistence risk in Minnesota in fall (September 1 –

November 30) and LPAI-affected counties (October 20 – November 14, 2018).

Table 13 County-level AIV environmental persistence risk reclassification scheme.

Majority value Risk category (value)

0 -2 Very Low / Low

3 Moderate

5 High

Figures 14 and 15 show commercial and backyard poultry operations in combination with 

counties having high risk for AIV persistence in the environment. Counties in the central portion 

of the state with the highest number of commercial operations, do not have the highest levels 

of AIV environmental persistence. Likewise, the highest concentrations of backyard operations 

are also in the central portions of the state where counties have lower risk of AIV persistence in 

the environment.
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Figure 14 Number of commercial poultry operations, counties with high fall season AIV environmental 

persistence risk, and LPAI-affected counties (October 20 – November 14, 2018).
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Figure 15 Number of backyard poultry operations, counties with high fall season AIV environmental 

persistence risk, and LPAI-affected counties (October 20 – November 14, 2018).

Using the majority statistic to aggregate risk to the county level has limitations and may not 

accurately capture risk at the poultry operation level. In most Minnesota counties, the 1km 

model shows diversity; i.e., within a given county, AIV environmental persistence risk ranges 

from low to very high. To better understand risk at the operation level, the diversity and range 

of 1km risk was evaluated within 3km and 10km buffers surrounding each of the recent 

Minnesota LPAI-infected operations. These buffer distances correspond to the typical zones 

used for emergency response and surveillance. 

This additional evaluation of diversity in buffers illustrates that even in low risk counties, an 

individual poultry operation may be in close proximity to moderate and high risk geographic 

areas at 1km spatial resolution. For example, as illustrated in Figure 16, both 3km and 10km 

buffer zones around an operation include moderate to very high risk 1km cells. Majority risk, at 

the county level in which this operation is located, is low. When evaluating the risk of AIV 

persistence in the environment, scale should be considered. Where feasible, it may be more 

applicable to use the higher resolution risk model (1km) to evaluate risk of environmental 

persistence near individual poultry operations.
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Figure 16 3km and 10km buffer zones around an individual commercial poultry operation (not shown) 

in the recent Minnesota LPAI outbreak (October 20 – November 14, 2018). Buffers are overlaid with 

the 1km AIV environmental persistence risk model.
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IX. Acknowledgements

We greatly appreciate the cooperation and support of the poultry industry and farm owners for 

allowing us access to their properties, for providing information on their biosecurity and 

production practices, and for their full cooperation with this investigation. 

We thank Peter Bonney and Sasidhar Malladi from the University of Minnesota for modeling 

support. We also thank Michelle Kromm, Jill Nezworski, Megan Lighty, Dale Lauer, Shauna Voss, 

Mary Donahue, and Stacy Pollock for assistance in obtaining diagnostic testing data to support 

model development. We thank Erica Spackman for providing experimental infection data.

This report would not have been possible without a large group of researchers, epidemiologists, 

laboratory staff, state animal health officials and data scientists who participated in the various 

analyses, drafting, and review of this report. We appreciate their dedication and professionalism 

in providing the best information possible to help producers reduce the risk of IAV introduction 

into their operations. 
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Appendix A: Case Series Questionnaire

 
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 Appendix B: Model Parameter Estimation

Barn-level LPAI diagnostic test data

Tables 14-21 give the date of testing, test type, surveillance protocol, and test result for the 

LPAI-infected barns on eight turkey meat-type premises in Minnesota. Stearns 1/Barn 2 and 

Stearns 4/ Barn 1 diagnostic test data are given in the main text.

Table 14 Summary of surveillance protocols and test results from the LPAI infected barns on Kandiyohi 

1.

Kandiyohi 1

Barn 1

Date Test type Sample7 Test result

Average (+) Ct 

value/ELISA 

titer

10/19/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/24/2018 rRT-PCR 1 pooled sample 1/1 positive 27

10/24/2018 AGID 10 serum samples 0/10 positive NA

10/29/2018 rRT-PCR 3 pooled samples 3/3 positive 25

10/29/2018 rRT-PCR 10 individual swabs 10/10 positive 28

10/29/2018 AGID 10 serum samples 6/10 positive NA

10/30/2018 AGID 30 serum samples 21/30 positive NA

11/01/2018 rRT-PCR 3 pooled samples 1/3 positive 36

11/01/2018 rRT-PCR 10 individual swabs 9/10 positive 31

11/01/2018 AGID 30 serum samples 30/30 positive NA

11/05/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/05/2018 rRT-PCR 10 individual swabs 1/10 positive 34

11/08/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/08/2018 rRT-PCR 10 individual swabs 1/10 positive 39

11/12/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/19/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/26/2018 rRT-PCR 3 pooled samples 0/3 positive NA

House 2

10/19/2018 rRT-PCR 1 pooled sample 1/1 positive 19

 7 Pooled rRT-PCR samples consisted of either 10 or 11 oropharyngeal swabs.
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10/24/2018 rRT-PCR 1 pooled sample 1/1 positive 25

10/24/2018 AGID 10 serum samples 10/10 positive NA

10/29/2018 rRT-PCR 3 pooled samples 0/3 positive NA

10/29/2018 rRT-PCR 10 individual swabs 0/10 positive NA

10/29/2018 AGID 10 serum samples 10/10 positive NA

10/30/2018 AGID 30 serum samples 30/30 positive NA

11/01/2018 rRT-PCR 3 pooled samples 2/3 positive 37

11/01/2018 rRT-PCR 10 individual swabs 1/10 positive 37

11/05/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/05/2018 rRT-PCR 10 individual swabs 2/10 positive 38

11/08/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/08/2018 rRT-PCR 10 individual swabs 0/10 positive NA

11/12/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/19/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/26/2018 rRT-PCR 3 pooled samples 0/3 positive NA

House 3

10/19/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/24/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/24/2018 AGID 10 serum samples 0/10 positive NA

10/29/2018 rRT-PCR 3 pooled samples 3/3 positive 23

10/29/2018 rRT-PCR 10 individual swabs 10/10 positive 29

10/29/2018 AGID 10 serum samples 0/10 positive NA

10/30/2018 AGID 30 serum samples 0/30 positive NA

11/01/2018 rRT-PCR 3 pooled samples 3/3 positive 25

11/01/2018 rRT-PCR 10 individual swabs 10/10 positive 28

11/01/2018 AGID 30 serum samples 0/30 positive NA

11/05/2018 rRT-PCR 3 pooled samples 2/3 positive 37

11/05/2018 rRT-PCR 10 individual swabs 6/10 positive 31

11/05/2018 AGID 30 serum samples 30/30 positive NA
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11/08/2018 rRT-PCR 3 pooled samples 1/3 positive 39

11/08/2018 rRT-PCR 10 individual swabs 4/10 positive 36

11/12/2018 rRT-PCR 3 pooled samples 1/3 positive 38

11/19/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/26/2018 rRT-PCR 3 pooled samples 0/3 positive NA

House 4

10/19/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/24/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/24/2018 AGID 10 serum samples 0/10 positive NA

10/29/2018 rRT-PCR 3 pooled samples 3/3 positive 25

10/29/2018 rRT-PCR 10 individual swabs 10/10 positive 29

10/29/2018 AGID 10 serum samples 0/10 positive NA

10/30/2018 AGID 30 serum samples 0/30 positive NA

11/01/2018 rRT-PCR 3 pooled samples 3/3 positive 30

11/01/2018 rRT-PCR 10 individual swabs 10/10 positive 31

11/01/2018 AGID 30 serum samples 17/30 positive NA

11/05/2018 rRT-PCR 3 pooled samples 1/3 positive 38

11/05/2018 rRT-PCR 10 individual swabs 5/10 positive 35

11/05/2018 AGID 30 serum samples 30/30 positive NA

11/08/2018 rRT-PCR 3 pooled samples 1/3 positive 39

11/08/2018 rRT-PCR 10 individual swabs 0/10 positive NA

11/12/2018 rRT-PCR 3 pooled samples 2/3 positive 38

11/19/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/26/2018 rRT-PCR 3 pooled samples 0/3 positive NA
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Table 15 Summary of surveillance protocols and test results from the LPAI infected barns on Kandiyohi 

2.

Kandiyohi 2

Barn 1

Date Test type Sample8 Result

Average (+) Ct 

value/ELISA 

titer

10/20/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/25/2018 AGID 9 serum samples 0/9 positive NA

11/01/2018 rRT-PCR 3 pooled samples 2/3 positive 24

11/05/2018 rRT-PCR 1 pooled sample 1/1 positive 33

11/08/2018 ELISA 10 serum samples 10/10 positive 1741

11/12/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/19/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/03/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 2

10/20/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/25/2018 AGID 10 serum samples 0/10 positive NA

11/01/2018 rRT-PCR 3 pooled samples 3/3 positive 32

11/05/2018 rRT-PCR 1 pooled sample 1/1 positive 22

11/08/2018 ELISA 10 serum samples 1/10 positive 347

11/12/2018 rRT-PCR 1 pooled sample 1/1 positive 35

11/16/2018 ELISA 10 serum samples 10/10 positive 4197

11/19/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/26/2018 rRT-PCR 3 pooled samples 0/3 positive NA

12/03/2018 rRT-PCR 1 pooled sample 0/1 positive NA

Barn 3

10/20/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/25/2018 AGID 9 serum samples 0/9 positive NA

11/01/2018 rRT-PCR 3 pooled samples 3/3 positive 37

 8 Pooled rRT-PCR samples consisted of either 10 or 11 oropharyngeal swabs.
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11/05/2018 rRT-PCR 1 pooled sample 1/1 positive 21

11/08/2018 ELISA 10 serum samples 0/10 positive NA

11/12/2018 rRT-PCR 1 pooled sample 1/1 positive 32

11/15/2018 ELISA 10 serum samples 10/10 positive 3758

11/19/2018 rRT-PCR 3 pooled samples 1/3 positive 39

11/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/03/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 4

10/20/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/25/2018 AGID 9 serum samples 0/9 positive NA

11/01/2018 rRT-PCR 3 pooled samples 1/3 positive 37

11/05/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/08/2018 ELISA 10 serum samples 1/10 positive 356

11/12/2018 rRT-PCR 1 pooled sample 1/1 positive 21

11/15/2018 ELISA 10 serum samples 0/10 positive NA

11/19/2018 rRT-PCR 3 pooled samples 3/3 positive 29

11/26/2018 rRT-PCR 1 pooled sample 1/1 positive 41

11/29/2018 ELISA 10 serum samples 10/10 positive 2237

12/03/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 5

10/20/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/25/2018 AGID 10 serum samples 0/10 positive NA

11/01/2018 rRT-PCR 3 pooled samples 3/3 positive 23

11/05/2018 rRT-PCR 1 pooled sample 1/1 positive 34

11/08/2018 ELISA 10 serum samples 10/10 positive 1478

11/12/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/19/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/03/2018 rRT-PCR 3 pooled samples 0/3 positive NA
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Table 16 Summary of surveillance protocols and test results from the LPAI infected barns on Kandiyohi 

3.

Kandiyohi 3

Barn 1

Date Test type Sample9 Test result

Average (+) Ct 

value/ELISA 

titer

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/26/2018 AGID 10 serum samples 0/10 positive NA

11/02/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/09/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 rRT-PCR 1 pooled sample 1/1 positive 38

11/15/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 rRT-PCR 1 pooled sample 1/1 positive 26

11/26/2018 rRT-PCR 1 pooled sample 1/1 positive 37

11/30/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/07/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/13/2018 ELISA 10 serum samples 10/10 positive 2095

12/14/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/21/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 2

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/26/2018 AGID 10 serum samples 0/10 positive NA

11/02/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/09/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 rRT-PCR 1 pooled sample 1/1 positive 28

11/26/2018 rRT-PCR 1 pooled sample 1/1 positive 25

 9 Pooled rRT-PCR samples consisted of either 10 or 11 oropharyngeal swabs.
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11/30/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/07/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/13/2018 ELISA 10 serum samples 10/10 positive 2779

12/14/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/21/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 3

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/26/2018 AGID 10 serum samples 0/10 positive NA

11/02/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/09/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 rRT-PCR 1 pooled sample 1/1 positive 36

11/15/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 rRT-PCR 1 pooled sample 1/1 positive 27

11/26/2018 rRT-PCR 1 pooled sample 1/1 positive 26

11/30/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/07/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/13/2018 ELISA 10 serum samples 10/10 positive 3777

12/14/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/21/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 4

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/26/2018 AGID 10 serum samples 0/10 positive NA

11/02/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/09/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 rRT-PCR 1 pooled sample 1/1 positive 24
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11/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/30/2018 rRT-PCR 1 pooled sample 1/1 positive 38

12/07/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/13/2018 ELISA 10 serum samples 10/10 positive 3513

12/14/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/21/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 5

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/26/2018 AGID 10 serum samples 0/10 positive NA

11/02/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/09/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 rRT-PCR 1 pooled sample 1/1 positive 22

11/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/30/2018 rRT-PCR 1 pooled sample 1/1 positive 33

12/07/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/13/2018 ELISA 10 serum samples 10/10 positive 3527

12/14/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/21/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 6

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/26/2018 AGID 10 serum samples 0/10 positive NA

11/02/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/09/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 ELISA 10 serum samples 0/10 positive NA
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11/21/2018 rRT-PCR 1 pooled sample 1/1 positive 27

11/26/2018 rRT-PCR 1 pooled sample 1/1 positive 39

11/30/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/07/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/13/2018 ELISA 10 serum samples 10/10 positive 2937

12/14/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/21/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 7

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/26/2018 AGID 10 serum samples 0/10 positive NA

11/02/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/09/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 rRT-PCR 1 pooled sample 1/1 positive 36

11/15/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 rRT-PCR 1 pooled sample 1/1 positive 27

11/26/2018 rRT-PCR 1 pooled sample 1/1 positive 37

11/30/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/07/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/13/2018 ELISA 10 serum samples 10/10 positive 2927

12/14/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/21/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 8

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/26/2018 AGID 10 serum samples 0/10 positive NA

11/02/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/09/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/12/2018 rRT-PCR 3 pooled samples 3/3 positive 28

11/15/2018 rRT-PCR 1 pooled sample 1/1 positive 27
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11/15/2018 ELISA 10 serum samples 0/10 positive NA

11/21/2018 ELISA 10 serum samples 10/10 positive 1318

11/21/2018 rRT-PCR 1 pooled sample 1/1 positive 35

11/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/30/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/07/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/13/2018 ELISA 10 serum samples 10/10 positive 3660

12/14/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/21/2018 rRT-PCR 3 pooled samples 0/3 positive NA
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Table 17. Summary of surveillance protocols and test results from the LPAI infected barns on Kandiyohi 

4.

Kandiyohi 4

Barn 1

Date Test type Sample10 Result

Average (+) Ct 

value/ELISA 

titer

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/23/2018 AGID 8 serum samples 0/8 positive NA

10/31/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/13/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/20/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/04/2018 rRT-PCR 1 pooled sample 1/1 positive 23

12/11/2018 rRT-PCR 1 pooled sample 1/1 positive 36

12/18/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/20/2018 ELISA 10 serum samples 10/10 positive 1465

12/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

1/03/2019 rRT-PCR 3 pooled samples 0/3 positive NA

1/07/2019 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 2

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/23/2018 AGID 10 serum samples 0/10 positive NA

10/31/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/13/2018 rRT-PCR 1 pooled sample 1/1 positive 25

11/20/2018 rRT-PCR 1 pooled sample 1/1 positive 22

11/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/04/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/11/2018 rRT-PCR 1 pooled sample 1/1 positive 38

12/18/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/20/2018 ELISA 9 serum samples 9/9 positive 3146

 10 Pooled rRT-PCR samples consisted of either 10 or 11 oropharyngeal swabs.
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12/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

1/03/2019 rRT-PCR 3 pooled samples 0/3 positive NA

1/07/2019 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 3

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/23/2018 AGID 10 serum samples 0/10 positive NA

10/31/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/13/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/20/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/26/2018 rRT-PCR 1 pooled sample 1/1 positive 20

12/04/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/11/2018 rRT-PCR 1 pooled sample 1/1 positive 37

12/18/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/20/2018 ELISA 10 serum samples 9/10 positive 1896

12/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

1/03/2019 rRT-PCR 3 pooled samples 0/3 positive NA

1/07/2019 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 4

10/22/2018 rRT-PCR 1 pooled sample 0/1 positive NA

10/23/2018 AGID 9 serum samples 0/9 positive NA

10/31/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/13/2018 rRT-PCR 1 pooled sample 1/1 positive 38

11/20/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/04/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/11/2018 rRT-PCR 1 pooled sample 1/1 positive 21

12/18/2018 rRT-PCR 1 pooled sample 1/1 positive 31

12/20/2018 ELISA 10 serum samples 10/10 

positive 1911

12/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA
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1/03/2019 rRT-PCR 3 pooled samples 0/3 positive NA

1/07/2019 rRT-PCR 3 pooled samples 0/3 positive NA

Table 18 Summary of surveillance protocols and test results from LPAI infected barns on Stearns 1. The 

testing summary for Barn 2 is included in the main report.

Stearns 1

Barn 1

Date Test type Sample11 Result

Average (+) Ct 

value/ELISA 

titer

10/30/2018 rRT-PCR 2 pooled samples 2/2 positive 20

10/30/2018 AGID 10 serum samples 10/10 positive NA

11/05/2018 rRT-PCR 1 pooled sample 1/1 positive 36

11/08/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/08/2018 ELISA 10 serum samples 10/10 positive 2465

11/12/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 3

10/30/2018 rRT-PCR 2 pooled samples 2/2 positive 36

10/30/2018 AGID 10 serum samples 10/10 positive NA

11/05/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/08/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/08/2018 ELISA 10 serum samples 10/10 positive 3251

11/12/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Table 19 Summary of surveillance protocols and test results from the LPAI infected barn on Stearns 2.

Stearns 2

Barn 1

Date Test type Sample12 Result

Average (+) Ct 

value/ELISA 

titer

10/31/2018 rRT-PCR 1 pooled sample 1/1 positive 23

10/31/2018 ELISA 10 serum samples 10/10 positive 1723

 11 Pooled rRT-PCR samples consisted of either 10 or 11 oropharyngeal swabs. 12 Pooled rRT-PCR samples consisted of either 10 or 11 oropharyngeal swabs.
Epidemiologic and Other Analyses of LPAI Affected Poultry Flocks February 1, 2019

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11/01/2018 rRT-PCR 1 pooled sample 1/1 positive 24

11/06/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/07/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/12/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/12/2018 ELISA 20 serum samples 19/20 positive 2134

Table 20 Summary of surveillance protocols and test results from the LPAI infected barns on Stearns 3.

Stearns 3

Barn 1

Date Test type Sample13 Test result

Average (+) Ct 

value/ELISA 

titer

11/01/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/02/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/08/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 rRT-PCR 1 pooled sample 1/1 positive 27

11/16/2018 ELISA 10 serum samples 0/10 positive NA

11/20/2018 rRT-PCR 1 pooled sample 1/1 positive 23

11/20/2018 ELISA 10 serum samples 10/10 positive 4867

11/27/2018 rRT-PCR 1 pooled sample 1/1 positive 37

11/28/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/03/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 2

11/01/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/02/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/08/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/15/2018 rRT-PCR 1 pooled sample 1/1 positive 28

11/16/2018 ELISA 10 serum samples 0/10 positive NA

11/20/2018 rRT-PCR 1 pooled sample 1/1 positive 21

11/20/2018 ELISA 10 serum samples 10/10 positive 4794

 13 Pooled rRT-PCR samples consisted of either 10 or 11 oropharyngeal swabs.
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11/27/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/28/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/03/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 3

11/01/2018 rRT-PCR 1 pooled sample 1/1 positive 37

11/01/2018 ELISA 20 serum samples 0/20 positive NA

11/02/2018 rRT-PCR 3 pooled samples 3/3 positive 24

11/08/2018 rRT-PCR 1 pooled sample 1/1 positive 28

11/15/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/16/2018 ELISA 10 serum samples 10/10 positive 2744

11/20/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/20/2018 ELISA 10 serum samples 10/10 positive 6812

11/26/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/28/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/03/2018 rRT-PCR 3 pooled samples 0/3 positive NA

Barn 4

11/01/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/02/2018 rRT-PCR 3 pooled samples 0/3 positive NA

11/08/2018 rRT-PCR 1 pooled sample 1/1 positive 26

11/15/2018 rRT-PCR 1 pooled sample 1/1 positive 27

11/16/2018 ELISA 10 serum samples 8/10 positive 793

11/20/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/20/2018 ELISA 10 serum samples 10/10 positive 4952

11/27/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/28/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/03/2018 rRT-PCR 3 pooled samples 0/3 positive NA
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Table 21 Summary of surveillance protocols and test results from LPAI infected barns on Stearns 4. The 

testing summary for Barn 1 is provided in the main report.

Stearns 4

Barn 2

Date Test type Sample14 Result

Average (+) Ct 

value/ELISA 

titer

11/02/2018 rRT-PCR 1 pooled sample 0/1 positive NA

11/06/2018 ELISA 10 serum samples 0/10 positive NA

11/14/2018 rRT-PCR 1 pooled sample 1/1 positive 21

11/19/2018 rRT-PCR 1 pooled sample 1/1 positive 22

11/19/2018 ELISA 10 serum samples 9/10 positive 1512

11/26/2018 rRT-PCR 1 pooled sample 0/1 positive NA

12/03/2018 rRT-PCR 3 pooled samples 0/1 positive NA

House 3

11/02/2018 rRT-PCR 2 pooled samples 0/2 positive NA

11/06/2018 ELISA 10 serum samples 0/10 positive NA

11/14/2018 rRT-PCR 1 pooled sample 1/1 positive 23

11/19/2018 rRT-PCR 1 pooled sample 1/1 positive 20

11/19/2018 ELISA 10 serum samples 0/10 positive NA

11/26/2018 rRT-PCR 1 pooled sample 1/1 positive 38

12/03/2018 rRT-PCR 3 pooled samples 0/1 positive NA

House 4

11/02/2018 rRT-PCR 2 pooled samples 2/2 positive 27

11/06/2018 ELISA 10 serum samples 0/10 positive NA

11/12/2018 rRT-PCR 1 pooled sample 1/1 positive 35

11/12/2018 ELISA 10 serum samples 10/10 positive 3298

11/19/2018 rRT-PCR 1 pooled sample 1/1 positive 36

11/26/2018 rRT-PCR 1 pooled sample 1/1 positive 38

12/03/2018 rRT-PCR 3 pooled samples 0/1 positive NA

 14 Pooled rRT-PCR samples consisted of either 10 or 11 oropharyngeal swabs.
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Sensitivity Analysis: Number of Swabs in rRT-PCR Pooled Samples

A sensitivity analysis was performed for the number of swabs included in the pooled 

samples tested by rRT-PCR. During the outbreak, pooled samples consisted of either 10 or 11 

oropharyngeal swabs, though it was often unclear in the data which pool size was used. The 

baseline pool size used in this analysis was 11 swabs. For the sensitivity analysis the time of virus 

introduction was estimated for the barns on Kandiyohi 4 and Stearns 3 assuming each rRT-PCR 

pooled sample consisted of 10 swabs. The comparison of the estimated time of virus 

introduction assuming rRT-PCR sample pool sizes of 10 and 11 swabs for Kandiyohi 4 and 

Stearns 3 are given in Table 22. The estimates for the time of virus introduction were highly 

similar between the two rRT-PCR pool size scenarios, which suggests results are robust to 

changes in the number of swabs per pooled sample.

Table 22 Most likely date of introduction (95% CI) for the barns that tested positive on Stearns 3 and 

Kandiyohi 4 during the 2018 H5N2 outbreak in Minnesota considering rRT-PCR pooled samples of 

size 11 and 10 swabs.

Premises Barn

Most likely date of virus 

introduction (95% CI):

11 swabs per rRT-PCR sample

Most likely date of virus 

introduction (95% CI):

10 swabs per rRT-PCR sample

Stearns 3

Barn 3 October 26 (Oct 19 – Oct 28) October 26 (Oct 19 – Oct 28)

Barn 4 October 31 (Oct 27 – Nov 4) October 31 (Oct 25 – Nov 3)

Barn 1 November 8 (Nov 2- Nov 10) November 7 (Oct 31 – Nov 9)

Barn 2 November 8 (Nov 2 – Nov 10) November 7 (Oct 31 – Nov 9)

Kandiyohi 4

Barn 2 October 28 (Oct 19 – Nov 8) October 27 (Oct 19 – Nov 8)

Barn 4 November 6 (Oct 23 – Nov 11) November 5 (Oct 23 – Nov 11)

Barn 3 November 19 (Nov 10 – Nov 22) November 18 (Nov 7 – Nov 22)

Barn 1 November 23 (Nov 15 – Nov 29) November 23 (Nov 13 – Nov 28)

Stochastic Disease Transmission Model Details

The transmission model used to simulate the spread of LPAI within a turkey house was a 

discrete stochastic individual-based transmission model where the infection status of individual 

birds was tracked at discrete simulation time steps. The disease states included susceptible, 

latently infected, infectious, removed/recovered, and seroconverted. The number of birds 

transitioning from the susceptible to the latently infected state at the beginning of a time step 

was simulated from a binomial distribution, where the probability of infection was dependent 

on the adequate contact rate, and proportion of infectious and alive birds. Once infected, the 

number of time steps each bird would be in the latently infected and then infectious state was 

simulated from distributions modeling the length of the latent and infectious periods. The 

number of time steps until the bird seroconverted following infection was also simulated from a 

distribution. For more details, see Bonney et al. (2018). The parameter estimates for the 

transmission model are given in Table 23.
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Table 23 Parameter estimates for the LPAI transmission model for turkey barns.

Parameter name Parameter description Distribution/Value

Contact rate

The mean number of direct or indirect 

contacts a bird has that are sufficient to 

transmit infection per unit time

Grid of 0.5 to 10.0 contacts 

per day discretized at 0.1 

intervals

Latent period 

distribution Length of the latent period

Gamma distribution (shape 

2.5839130, scale 0.2441542; 

mean 0.63 days)

Infectious period 

distribution Length of the infectious period

Gamma distribution (shape 

4.040337, scale 2.915678; 

mean 11.78 days)

Time to 

seroconversion Time to seroconvert post-infection

Gamma distribution (shape 

3.556011, scale 1.629968; 

mean 5.80 days)

Proportion 

seroconverting

Proportion of LPAI infected turkeys that 

seroconvert

0.99

Estimation of transmission model parameters 

There is considerable uncertainty in the within-house adequate contact rate for LPAI in turkeys, 

a parameter that determines the rate of infection spread in the transmission model. Saenz et al. 

(2012) was the only study identified where a contact rate was estimated for LPAI in turkeys (11). 

The mean contact rate estimated in Saenz et al. (2012) was 2.01 (95% CI: 1.6-2.5) from LPAI 

H7N1 turkey inoculation data. The contact rate can vary depending on factors such as housing, 

bird species, and LPAI strain. In light of this uncertainty, a fairly wide range of candidate contact 

rate values were used, ranging from 0.5 to 10.0 contacts per day.

Parameters for the length of the latent and infectious period were estimated using a maximum 

likelihood method from experimental data in which turkeys were inoculated with H5 or H7 LPAI 

strains. Both distributions were assumed to be Gamma distributed. For details on the maximum 

likelihood method see Appendix 2 of “An Assessment of the Risk Associated with the Movement 

of Turkey Hatching Eggs Into, Within, and Out of a Control Area during a Highly Pathogenic Avian 

Influenza Outbreak”(USDA:APHIS:VS, 2015). Insufficient data was available for H5 LPAI strains 

leading to both H5 and H7 LPAI strains being included in the estimation of the distributions.

The inoculation studies used in the estimation of the latent period distribution include Pillai et 

al. (2010), Iqbal et al. (2012), and Pantin-Jackwood et al. (2017). Only the turkeys inoculated 

with H5 LPAI strains isolated from infected farms were used from Pillai et al. (2010), as the 

strains from wild birds and live bird markets appeared to be poorly adapted to turkeys. The data 

from Iqbal et al. (2012) consisted of 20 turkeys, specifically turkeys 81-90 from Table 8 in the 

article and turkeys 21-30 from Table S1 in the article, that were infected with LPAI H7N1. The 

data from Pantin-Jackwood et al. (2017) consisted of 17 turkeys inoculated with a 106

EID50 dose 

of LPAI H7N1. The estimated shape parameter of the gamma distribution for the length of the 

latent period was 2.5839130, while the estimated scale parameter was 0.2441542. The mean of 

the distribution is 0.63 days. 
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The inoculation studies used in the estimation of the infectious period distribution include Pillai 

et al. (2010), Saenz et al. (2012), Iqbal et al. (2012), Comin et al. (2011), Pantin-Jackwood et al. 

(2017), and Spackman et al. (2010). Pillai et al. (2010) was the only study included with H5 data. 

As in the estimation of the latent period distribution, the only Pillai et al. (2010) data used was 

from those turkeys inoculated with strains from infected farms. In Saenz et al. (2012), the data 

consisted of two separate transmission experiments, one involving 1 turkey inoculated with LPAI 

H7N1 and 40 contact turkeys, and the other involving 1 inoculated turkey and 41 contact 

turkeys (Table 5 and Table 6 in the article). The data used from Iqbal et al. (2012) consisted of 

the 10 turkeys inoculated with LPAI H7N1 and 10 contact turkeys given in Table 8 of the article. 

Similarly, turkeys k10-18 inoculated with LPAI H7N3 from Table 3 in Comin et al. (2011) and five 

turkeys inoculated with a 104

EID50 dose of LPAI H7N1 along with the 17 turkeys inoculated with 

a 106

EID50 dose of LPAI H7N8 from Pantin-Jackwood et al. (2017) were used in the estimation of 

the infectious period. Lastly, data on 113 turkeys inoculated with 12 different H7 strains were 

included from Spackman et al. (2010). The estimated parameters of the gamma distribution for 

the length of the infectious period were a shape equal to 4.040337 and scale equal to 2.915678. 

The mean of the distribution is 11.78 days.

Parameters for the distribution for the time from infection until seroconversion, assumed to be 

gamma distributed, were estimated using the same maximum likelihood method employed in 

the estimation of the infectious and latent period distribution parameters. Usable data for the 

estimation of the time to seroconversion was scarce, leading to data from inoculation studies 

involving any LPAI strain being included. The studies used were Dundon et al. (2007), Morales 

(2008), Homme et al. (1970), and Preskenis (2010), which involved H4, H6, H7, and H9 LPAI 

strains. Only in Dundon et al. (2007) were serum samples taken within 7 days post inoculation. 

Samples in the other three studies were taken at 7 day increments, which introduces a fair 

amount of uncertainty into the time to seroconversion distribution. The estimated shape

parameter of the distribution was 3.556011 and scale was 1.629968. The mean of the 

distribution is 5.80 days. The proportion of turkeys that seroconvert was based on Spackman et 

al. (2010) in which 88/89 (99%) turkeys inoculated with twelve North American H7 LPAI virus 

isolates had detectable antibody by day 18 to 21 post-inoculation. For comparison, 110/116 

(~95%) of the surviving chickens had detectable antibody in the same study.
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Appendix C: Data Factors and Processing for Geospatial Analysis 

Predicting Avian Influenza Virus Persistence in the Environment

Data Preparation

All data were prepared using tools within ArcGIS 10.6, and final layers were projected to 

Universal Transverse Mercator (UTM) Zone 15 North in the datum World Geodetic System 1984 

(WGS1984).

Predictive Factors and Data Sources: 

• Water Presence

Avian Influenza Viruses (AIV) have been shown to have improved survival in water 

compared to dry land (Brown et al., 2009; USGS, 2011). United States Geological Survey 

(USGS) Gap Analysis Project hydrography data collected between 1994 and 2004, 

derived from satellite imagery at a 30m resolution, were downloaded and reclassified 

into two categories of surface water based on suitability for AIV survival: presence of 

fresh water (high suitability), or no water present (low suitability). The surface water 

presence layer was aggregated to a one-kilometer (1km) resolution. 

• Water Temperature 

Water temperature is inversely associated with the rate of AIV survival (Brown et al., 

2009; Keeler et al., 2012; Lang et al., 2008; Stallknecht et al., 1990a; 1990b). USGS 

Moderate Resolution Imaging Spectroradiometer (MODIS)-derived 8-day land surface 

and emissivity scenes were downloaded for 2015-2017. Using R version 3.3.3, individual 

scenes were masked by quality indicators (i.e., cloud cover) and recombined to create 

summary temperature surfaces by season, at a spatial resolution of 1km (Grim and 

Knievel, 2013; Ke and Song, 2014; NASA, 2012). Refined MODIS data were then masked 

with the water presence layer to reflect locations only where surface water was present, 

and reclassified to represent high suitability (< 10° C), moderate suitability (≥ 10°C and < 

20°C), or low suitability (≥ 20°C) for AIV survival (Brown et al., 2007; Brown et al., 2009; 

Keeler et al., 2014; Nazir et al., 2010). 

• Wetlands and Wildlife Refuges 

U.S. Fish and Wildlife Service (USFWS) National Wetland Inventory data were used to 

identify locations of wetlands and wildlife refuges, which are considered favorable for 

AIV persistence (USFWS, 2016; Keeler et al., 2012; Belkhiria, et al., 2016; Iglesias et al., 

2010; Fuller et al., 2010). Locations classified as ‘freshwater emergent wetland’ and 

‘freshwater forested/shrub wetland’ were extracted; data were reclassified based on 

presence or absence of either wetland type and resampled to 1km. For wildlife refuges, 

USFWS Cadastral data were obtained and reclassified at a 1km resolution based on 

presence or absence of National Wildlife Refuge (NWR) land. 

• Wild Bird Presence 

Data for wild bird presence were provided by the USGS Patuxent Wildlife Research 

Center. These data were derived using a spatio-temporal model estimating seasonal 

occurrence (presence) and abundance of 10 dabbling duck species (Table 15) 

throughout the conterminous United States at a 1km resolution (Humphreys, 2019). The 
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occurrence model data for each species was combined then reclassified to create a 

presence/absence dataset for all 10 dabbling duck species in Minnesota. 

Table 24 Common and scientific names of dabbling duck species that were included in the wild bird 

presence model.

Common Name Scientific Name

American black duck Anas rubripes

American wigeon Anas americana

Blue-winged teal Anas discors

Cinnamon teal Anas cyanoptera

Gadwall Anas strepera

Green-winged teal Anas carolinensis

Mallard Anas platyrhynchos

Mottled duck Anas fulvigula

Northern pintail Anas acuta

Northern shoveler Anas clypeata

• Poultry Operations 

Poultry operation data were provided by the Minnesota Board of Animal Health. The 

data included location information for 827 commercial operations and 8,581 backyard 

operations. These data were summarized by county to illustrate the number of 

commercial and backyard premises in relation to counties at high risk for AIV

introduction from wild birds.